RNA editing in kinetoplastid parasites entails massive remodeling of mitochondrial mRNAs by posttranscriptional insertion and deletion of uridine (U) residues to generate mRNAs encoding proteins involved in parasite bioenergetics. U insertion/deletion RNA editing is unique to kinetoplastids and essential for T. brucei survival and virulence. The RNA editing holoenzyme comprises two subcomplexes: RECC (RNA Editing Core Complex) and RESC (RNA Editing Substrate Binding Complex). Trans-acting guide RNAs (gRNAs) direct U insertion/deletion, acting sequentially such that editing proceeds 3' to 5' along an mRNA. RECC contains the editing enzymes, and the mechanisms by which RECC catalyzes U insertion/deletion at a single editing site have been extensively studied. More recently, RESC has emerged as the scaffold for editing and a coordinator of interactions between mRNAs, gRNAs, and RECC, although the mechanisms by which it accomplishes these tasks are essentially unknown. Adding even more complexity, editing of specific mRNAs is differentially regulated between human bloodstream form (BF) and insect vector procyclic form (PF) parasites. The mechanism(s) of developmental regulation, and the precise point(s) in the life cycle at which they are effected, remain entirely mysterious. The goals of this application are to (1) delineate the roles of distinct components of RESC and a novel RESC-related complex in gRNA trafficking and utilization, and (2) to define aspects of the editing process that serve as control points for stage specific regulation during the T. brucei life cycle. To accomplish these goals, we developed an innovative and powerful bioinformatic tool (HTS/TREAT) that permits us to now examine the mitochondrial transcriptome at single nucleotide resolution, and group partially edited mRNA intermediates in ways that illuminate specific aspects of gRNA usage. Combining this bioinformatic approach with RNA immunoprecipitation and protein-protein interaction studies, we will define the mechanisms by which specific RESC proteins function, correlate these to RESC heterogeneity and dynamic interactions, and elucidate the steps in the editing process that are developmentally controlled. We will also take advantage of recent technology that permits generation of substantial numbers of insect metacyclic form (MF) T. brucei, a largely unexplored intermediate between PF and BF. Using HTS/TREAT, we will define the patterns of RNA editing in MFs, and reveal whether BF RNA editing patterns for some or all mRNAs emerge in MFs, providing insight into the temporal control of RNA editing during T. brucei development. The proposed studies address several important gaps in knowledge regarding the essential kinetoplastid RNA editing process. Moreover, this work may help lay the foundation for future drug development and provide important insights into ribonucleoprotein complex function in higher organisms. ! ! !